Lipolytic enzyme LIP2

ABSTRACT

The present invention provides a novel nucleic acid sequence, designated LIP2, encoding a lipolytic enzyme and the corresponding encoded amino acid sequences. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding at least one novel lipolytic enzyme, recombinant lipolytic enzyme proteins and methods for producing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 10/555,560,filed Nov. 20, 2006, now abandoned, which is a U.S. National Phaseapplication of International Application No. PCT/US2004/014684, filedMay 12, 2004, which claims the benefit of U.S. Provisional ApplicationNo. 60/470,031, filed May 12, 2003, which are hereby incorporated intheir entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R.§1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “30802-2D1_ST25.txt” created onOct. 12, 2011, which is 8,133 bytes in size.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable.

FIELD OF THE INVENTION

The present invention relates to novel lipolytic enzymes andpolynucleotides encoding the lipolytic enzyme polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid constructs as well as methods forproducing and using the polypeptides.

BACKGROUND OF THE INVENTION

For a number of years lipolytic enzymes have been used as detergentenzymes, i.e. to remove lipid or fatty stains from clothes and othertextiles.

Lipolytic enzymes include, but are not limited to, lipases andesterases. Lipases are versatile biocatalysts that can performinnumerable different reactions. Unlike other hydrolases that work inaqueous phase, lipases are unique as they act at the oil/waterinterface. Besides being lipolytic, lipases also possess esterolyticactivity and thus have a wide substrate range.

A need exists for novel lipolytic enzymes having improved washing and/ordishwashing properties, and the object of the present invention is toprepare such enzymes

Although lipolytic compositions have been previously described, thereremains a need for new and improved lipolytic compositions for use inhousehold detergents, or laundry detergents, etc. Lipases that exhibitimproved performance are of particular interest.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a polypeptide havinglipolytic activity, designated herein as LIP2. In some embodiments, thepresent invention relates to a substantially pure polypeptide orlipolytic enzyme having lipolytic activity, wherein said polypeptide orlipolytic enzyme comprises an amino acid sequence having at least 75%,at least 85%, at least 95% amino acid sequence identity with an aminoacid sequence set forth in SEQ ID NO.:3, wherein said polypeptide haslipolytic activity. In other embodiments, the polypeptide comprises anamino acid sequence having at least 75% sequence identity with an LIP2amino acid sequence set forth in SEQ ID NO.:3 and is encoded by apolynucleotide having a nucleic acid sequence having at least 75%, atleast 85%, or at least 95% nucleic acid sequence identity with a nucleicacid sequence set forth in SEQ ID NO.:1. In another embodiment, thepolynucleotide or lipolytic enzyme is derived from Bacillus. In anotherembodiment, the polynucleotide encoding a lipolytic enzyme is shown inFIG. 3.

In second aspect the present invention relates to a polynucleotideencoding a lipolytic polypeptide or enzyme. In one embodiment, thepolynucleotide encodes a lipolytic polypeptide or enzyme is derived fromBacillus. In other embodiments, the polynucleotide encodes an amino acidsequence having at least 75% sequence identity with an LIP2 amino acidsequence set forth in SEQ ID NO.:3 and has a nucleic acid sequencehaving at least 75%, at least 85%, or at least 95% nucleic acid sequenceidentity with a nucleic acid sequence set forth in SEQ ID NO.:1. Inanother embodiment, the polynucleotide encodes a lipolytic polypeptideor enzyme having an amino acid sequence shown in FIG. 3.

In a third aspect the present invention relates to a nucleic acidconstruct comprising the nucleotide sequence, which encodes for aninventive lipolytic polypeptide or enzyme, operably linked to one ormore control sequences that direct the production of the lipolyticpolynucleotide or enzyme in a suitable host.

In a fourth aspect the present invention relates to a recombinantexpression vector comprising the nucleic acid construct of theinvention.

In a fifth aspect the present invention relates to a recombinant hostcell comprising the nucleic acid construct of the invention.

In a sixth aspect the present invention relates to a method forproducing a lipolytic enzyme of the invention, the method comprising

-   -   a) transforming a host cell with a nucleic acid encoding an        inventive lipolytic polynucleotide or enzyme described herein;    -   b) culturing the host cell under conditions to produce the        polypeptide or enzyme; and    -   c) recovering the polypeptide or enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the genomic DNA sequence for LIP2. The coding sequence is inbold and underlined.

FIG. 2 is the cDNA sequence for LIP2.

FIG. 3 is the amino acid sequence for LIP2.

FIG. 4 is a table summarizing the details of the putative ORF's encodingthe inventive proteins with esterase/lipase activity.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents, patentapplications, articles and publications, including all sequencesdisclosed within such patents and publications, referred to herein areexpressly incorporated by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

All publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

I. Definitions

The terms “lipolytic polypeptides”, “lipolytic proteins”, “lipolyticenzymes” or “lipase enzymes” refer to a polypeptide, protein or enzymeexhibiting a lipid degrading capability such as a capability ofdegrading a triglyceride or a phospholipid. The lipolytic enzyme may be,for example, a lipase, a phospholipase, an esterase or a cutinase. Thephrase “esterase/lipase enzyme” may be used interchangeably herein.

For the present invention, lipolytic activity may be determinedaccording to any procedure known in the art. See, for example, Gupta etal, Biotechnol. Appl. Biochem. (2003) 37:63-71; Andre, Christophe, etal, U.S. Pat. No. 5,990,069 (International Publication WO 96/18729A1).

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between the nucleic acid sequence that encodes alipolytic polypeptide, lipolytic enzyme, lipase enzyme or the lipaseamino acid sequence, when aligned using a sequence alignment program.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 50, 55, 60, 65, 70, 75, 80,85, 90, 93, 95, 97, 98 and 99% or more sequence identity to a givensequence, e.g., the coding sequence for LIP2, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at www.ncbi.nlm.nih.gov/BLAST. Seealso, Altschul, et al., 1990 and Altschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, et al., 1997).

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

The term “derived” encompasses the terms originated from, obtained orobtainable from, and isolated from.

The term “nucleic acid” refers to DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”,“polynucleotide” or “nucleic acid molecule” may be used interchangeablyherein. Because the genetic code is degenerate, more than one codon maybe used to encode a particular amino acid, and the present invention.

The term “protein” refers to polymers of large molecular mass composedof one or more polypeptide chains and whose monomers are amino acidsjoined together by peptide bonds. The terms “protein” and “polypeptide”are sometimes used interchangeably herein. The conventional one-letteror three-letter code for amino acid residues is used herein.

The term “enzyme” refers to a protein having catalytic activity. Forexample, catalytic activity includes lipolytic activity. Theconventional one-letter or three-letter code for amino acid residues isused herein.

The term “host cell” refers to a cell that contains a vector andsupports the replication, and/or transcription or transcription andtranslation (expression) of the expression construct. Host cells for usein the present invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as yeast, plant, insect, amphibian, or mammaliancells. In a one embodiment according to the present invention, “hostcell” means the cells of the genus Bacillus. In another preferredembodiment according to the invention, “host cell” means the cells ofStreptomyces. A Streptomyces means any bacterial strain that is a memberof the genus Streptomyces as classified in Buchanan et al., The ShorterBergey's Manual For Determinative Bacteriology (Williams & Wilkens1982). Particularly preferred strains of Streptomyces include S.lividens, S. rubiginosus, and S. coelicolor. S. lividens is described inLomovskaya et al., J. Virology 9:258 (1972). However, one of skill willrealize that any appropriate host cell, e.g., bacterial, fungal,eukaryotic and plant cell may be used.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,underexpressed or not expressed at all. Also encompassed by the presentinvention is the overexpression of a native gene, possibility due to thepresence of additional copies of a native gene, or associating a nativegene with a promoter that is heterologous to the gene.

The term “secretory signal sequence” refers to a DNA sequence thatencodes a polypeptide (a “secretory peptide” or “secretory signalpeptide”) that, as a component of a larger polypeptide, directs thelarger polypeptide through a secretory pathway of a cell in which it issynthesized. The larger peptide is commonly cleaved to remove thesecretory peptide during transit through the secretory pathway to yieldthe secretory signal peptide and a smaller peptide commonly referred toas the mature polypeptide.

A “naturally occurring” composition is one produced by a naturallyoccurring source and which comprises one or more of the inventivelipolytic or lipase components wherein each of these components is foundin a proportion relative to other proteins produced by the source. Anaturally occurring composition is one that is produced by an organismunmodified with respect to the lipase enzymes such that the ratio of thecomponent enzymes is unaltered from that produced by the nativeorganism.

A “non-naturally occurring” composition encompasses those compositionsproduced by: (1) combining component lipase polypeptides or enzymeseither in a naturally occurring ratio or non-naturally occurring, i.e.,altered, ratio; or (2) modifying an organism to overexpress orunderexpress one or more lipolytic polypeptide or enzyme; or (3)modifying an organism such that at least one lipolytic polypeptide orenzyme is deleted or (4) modifying an organism to express a heterologouscomponent lipolytic polypeptide or enzyme.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. The promoter may bethe promoter normally associated with the downstream gene or it may beheterologous, i.e., from another gene or another microorganism as longas it function to direct the gene. Examples of suitable promoters fordirecting the transcription of the DNA sequence encoding a variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), e.g. as described in WO 93/10249 the promoters of the Bacillusstearothermophilus maltogenic amylase gene (amyM), the promoters of theBacillus amyloliquefaciens α-amylase (amyQ), the promoters of theBacillus subtilis xylA and xylB genes etc. A preferred promoter when thetransformation host cell is Bacillus is the aprE promoter. In one aspectthe promoter is an inducible promoter. In one aspect, when the host cellis a filamentous fungus, the promoter is the T. reesei cbh1 promoterwhich is deposited in GenBank under Accession Number D86235. In anotheraspect the promoter is a cbh II or xylanase promoter from T. reesei.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader, i.e., a signal peptide, is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

The terms “nucleic acid construct”, “DNA construct” or “DNA vector”refer to a nucleotide or nucleic acid sequence which comprises one ormore DNA fragments encoding the novel lipolytic polypeptide or enzyme.Included in “DNA vectors” are “expression vectors.” Typical expressionvectors contain regulatory sequences such as, transcription andtranslation terminators, transcription and translation initiationsequences, signal sequences, and promoters useful for regulation of theexpression of the particular nucleic acid. The term “promoter” is usedin its ordinary sense to refer to a polynucleotide sequence involved inthe control of the initiation of transcription of a polynucleotidesequence encoding a protein. A “signal sequence” refers to a signalpeptide or a portion of a protein that is capable of directing thetransport of a desired protein in bioactive form from a host. The matureform of an extracellular protein lacks the signal sequence which iscleaved off during the secretion process. While not meant to limit theinvention, the number of amino acid residues in a signal peptide may bebetween about 5 and about 100 amino acid residues. Signal sequence maybe modified to provide for cloning sites that allow for the ligation ofDNA or insertion of DNA encoding a lipolytic polypeptide or enzyme. Thevectors optionally comprise generic expression cassettes containing atleast one independent terminator sequence, sequences permittingreplication of the cassette in prokaryotes, eukaryotes, or both, (e.g.,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Giliman and Smith, Gene8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987); Berger andKimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY,VOL 152, Academic Press, Inc., San Diego, Calif. (“Berger”); Scheider,B., et al., Protein Expr. Purif 6435:10 (1995); Sambrook et al.MOLECULAR CLONING—A LABORATORY MANUAL (2ND ED.) VOL. 1-3, Cold SpringsHarbor Publishing (1989) (“Sambrook”); and CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Ausubel et al. (eds.), Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., (1997 Supplement) (“Ausubel”). Cloning vectors useful inStreptomyces are known and reference is made to U.S. Pat. Nos.4,338,397; 4,411,994; 4,513,085; 4,513,086; 4,745,056; 5,514,590; and5,622,866 and WO88/07079.

The term “gene” refers to the segment of DNA involved in producing apolypeptide chain or protein, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

The terms “heterologous” or “exogenous” with reference to apolynucleotide or protein refer to a polynucleotide or protein that doesnot naturally occur in a host cell. In some embodiments, the protein isa commercially important industrial protein. It is intended that theterm encompass proteins that are encoded by naturally occurring genes,mutated genes and/or synthetic genes. For example the nucleic acidsequence may comprise two or more subsequences that are not normallyfound in the same relationship to each other in nature. For instance,the nucleic acid is typically recombinantly produced, having two or moresequences, e.g., from unrelated genes arranged to make a new functionalnucleic acid, e.g., a promoter from one source and a coding region fromanother source. Similarly, a heterologous protein will often refer totwo or more subsequences that are not found in the same relationship toeach other in nature (e.g., a fusion protein).

The terms “homologous” or “endogenous” with reference to apolynucleotide, protein or enzyme refer to a polynucleotide, protein orenzyme that occurs naturally in the host cell.

The terms “isolated” or “purified” refer to a nucleic acid, amino acidor protein that is removed from at least one component with which it isnaturally associated.

The term “substantially pure polypeptide”, “substantially pure enzyme”refers to a polypeptide or enzyme preparation which contains at the most10% by weight of other polypeptide material with which it is nativelyassociated (lower percentages of other polypeptide material arepreferred, e.g. at the most 8% by weight, at the most 6% by weight, atthe most 5% by weight, at the most 4% at the most 3% by weight, at themost 2% by weight, at the most 1% by weight, and at the most ½% byweight). Thus, it is preferred that the substantially pure polypeptideis at least 92% pure, i.e. that the polypeptide constitutes at least 92%by weight of the total polypeptide material present in the preparation,and higher percentages are preferred such as at least 94% pure, at least95% pure, at least 96% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99%, and at the most 99.5% pure. Thepolypeptides disclosed herein are preferably in a substantially pureform. In particular, it is preferred that the polypeptides disclosedherein are in “essentially pure form”, i.e. that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods.Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form”.

In general, nucleic acid molecules which encode an inventive lipolyticpolypeptide or enzyme will hybridize, under moderate to high stringencyconditions to the sequence provided herein as SEQ ID NO:2 (cDNAsequences). However, in some cases a lipase-encoding nucleotide sequenceis employed that possesses a substantially different codon usage, whilethe protein encoded by the lipase-encoding nucleotide sequence has thesame or substantially the same amino acid sequence as the nativeprotein. For example, the coding sequence may be modified to facilitatefaster expression of an inventive lipase in a particular prokaryotic oreukaryotic expression system, in accordance with the frequency withwhich a particular codon is utilized by the host. Te'o, et al. (2000),for example, describes the optimization of genes for expression infilamentous fungi.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “moderate” or “intermediate stringency” at about 10-20° below the Tmof the probe; and “low stringency” at about 20-25° below the Tm.Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify sequences having about 80% or more sequence identity with theprobe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al., 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDSand 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

The terms “transformed”, “stably transformed” or “transgenic” withreference to a cell refers to a cell has a non-native (heterologous)nucleic acid sequence integrated into its genome or as an episomalplasmid that is maintained through multiple generations.

The term “expression” refers to the process by which a polypeptide isproduced based on the nucleic acid sequence of a gene. The processincludes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, includes “transfection”, or “transformation” or“transduction” and refers to the incorporation of a nucleic acidsequence into a eukaryotic or prokaryotic cell where the nucleic acidsequence may be incorporated into the genome of the cell (for example,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (for example, transfectedmRNA).

It follows that the terms “lipolytic polypeptide expression”, “lipolyticenzyme expression” and “lipase expression” refer to transcription andtranslation of an inventive lipolytic polypeptide, lipolytic enzyme orlipase gene, the products of which include precursor RNA, mRNA,polypeptide, post-translationally processed polypeptides. By way ofexample, assays for lipase expression include Western blot for a lipaseprotein, Northern blot analysis and reverse transcriptase polymerasechain reaction (RT-PCR) assays for lipase mRNA, and lipase activityassays as known in the art.

The term “surfactant” refers to any compound generally recognized in theart as having surface active qualities. Thus, for example, surfactantscomprise anionic, cationic and nonionic surfactants such as thosecommonly found in detergents. Anionic surfactants include linear orbranched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates havinglinear or branched alkyl groups or alkenyl groups; alkyl or alkenylsulfates; olefinsulfonates; and alkanesulfonates. Ampholytic surfactantsinclude quaternary ammonium salt sulfonates, and betaine-type ampholyticsurfactants. Such ampholytic surfactants have both the positive andnegative charged groups in the same molecule. Nonionic surfactants maycomprise polyoxyalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, fatty acid glycerinemonoesters, and the like.

The term “detergent composition” refers to a mixture which is intendedfor use in a wash medium for the laundering of soiled fabrics. In thecontext of the present invention, such compositions may include, inaddition to lipases and surfactants, additional enzymes (e.g.,hydrolytic, proteolytic, etc.), builders, bleaching agents, bleachactivators, bluing agents and fluorescent dyes, caking inhibitors,masking agents, activators, antioxidants, and solubilizers.

The terms “active” and “biologically active” refer to a biologicalactivity associated with a particular protein and are usedinterchangeably herein. For example, the enzymatic activity associatedwith a protease is proteolysis and, thus, an active protease hasproteolytic activity. It follows that the biological activity of a givenprotein refers to any biological activity typically attributed to thatprotein by those of skill in the art.

When employed in enzymatic solutions, the inventive lipolyticpolypeptide or enzyme component is generally added in an amountsufficient to allow the highest rate of fatty stain removal, which canbe readily determined by the skilled artisan. However, when employed,the weight percent of the lipolytic polypeptide or enzyme component isfrom preferably about 1, preferably about 5, preferably about 10,preferably about 15, or preferably about 20 weight percent to preferablyabout 25, preferably about 30, preferably about 35, preferably about 40,preferably about 45 or preferably about 50 weight percent. Furthermore,preferred ranges may be about 0.5 to about 15 weight percent, about 0.5to about 20 weight percent, from about 1 to about 10 weight percent,from about 1 to about 15 weight percent, from about 1 to about 20 weightpercent, from about 1 to about 25 weight percent, from about 5 to about20 weight percent, from about 5 to about 25 weight percent, from about 5to about 30 weight percent, from about 5 to about 35 weight percent,from about 5 to about 40 weight percent, from about 5 to about 45 weightpercent, from about 5 to about 50 weight percent, from about 10 to about20 weight percent, from about 10 to about 25 weight percent, from about10 to about 30 weight percent, from about 10 to about 35 weight percent,from about 10 to about 40 weight percent, from about 10 to about 45weight percent, from about 10 to about 50 weight percent, from about 15to about 20 weight percent, from about 15 to about 25 weight percent,from about 15 to about 30 weight percent, from about 15 to about 35weight percent, from about 15 to about 30 weight percent, from about 15to about 45 weight percent, from about 15 to about 50 weight percent.

II. Molecular Biology I

In one embodiment this invention provides for the expression oflipolytic polypeptide or enzyme genes under the control of a promoterfunctional in a host cell. Therefore, this invention relies on routinetechniques in the field of recombinant genetics. Basic texts disclosingthe general methods of use in this invention include Sambrook et al.,Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and Ausubel et al.,eds., Current Protocols in Molecular Biology (1994)).

In one embodiment, the polypeptide having lipolytic activity has atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%and 99% sequence identity with the LIP2 amino acid sequence set forth inSEQ ID NO: 3. In other embodiments, the polypeptide having lipolyticactivity comprises the amino acid sequence of SEQ ID NO: 3

In one embodiment, the polypeptide having lipolytic activity has atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%and 99% sequence identity with the LIP2 amino acid sequence set forth inSEQ ID NO: 3. In other embodiments, the polypeptide having lipolyticactivity comprises the amino acid sequence of SEQ ID NO: 3 or apolypeptide having lipolytic enzyme having at least 80% sequenceidentity with the sequence of SEQ ID NO: 3 is encoded by a nucleic acidsequence having at least 70%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99%nucleotide sequence identity with SEQ ID NO:1.

In one embodiment, the nucleotide sequence encoding a polypeptide havinglipolytic activity has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 93%, 95%, 97%, 98% and 99% sequence identity with the LIP2nucleotide sequence set forth in SEQ ID NO: 1. In other embodiments, thenucleic acid sequence encoding a polypeptide having lipolytic activitycomprises the LIP2 nucleic acid sequence of SEQ ID NO: 1

A polynucleotide (nucleic acid sequence) or polypeptide (amino acidsequence) having a certain percent (e.g., 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 93%, 95%, 97%, 98% or 99%) of sequence identity withanother sequence means that when aligned, that percent of bases or aminoacid residues are the same in comparing the two sequences. Thisalignment and the percent homology or identity can be determined usingany suitable software program known in the art, for example thosedescribed in Current Protocols in Molecular Biology (Ausubel et al., eds1987 Supplement 30, section 7.7.18). Preferred programs include GCGPileup program, FASTA and BLAST. Another preferred alignment program isALIGN Plus and TFASTA.

In one embodiment, the polynucleotide or polypeptide is derived fromBacillus.

A. Methods for Identifying Lipolytic Enzyme Genes

The invention, in one aspect, encompasses a nucleic acid moleculeencoding a lipolytic polypeptide or enzyme described herein. The nucleicacid may be a DNA molecule.

Techniques that can be used to isolate lipolytic polypeptide orenzyme-encoding DNA sequences are well known in the art and include, butare not limited to, cDNA and/or genomic library screening with ahomologous DNA probes and expression screening with activity assays orantibodies against a lipolytic polypeptide or enzyme. Any of thesemethods can be found in Sambrook, et al. or in CURRENT PROTOCOLS INMOLECULAR BIOLOGY, F. Ausubel, et al., ed. Greene Publishing andWiley-Interscience, New York (1987) (“Ausubel”).

The invention, in one aspect, encompasses a nucleic acid moleculeencoding a lipolytic polypeptide or enzyme described herein. The nucleicacid may be a DNA molecule.

Techniques that can be used to isolate lipolytic polypeptide orenzyme-encoding DNA sequences are well known in the art and include, butare not limited to, cDNA and/or genomic library screening with ahomologous DNA probes and expression screening with activity assays orantibodies against a lipolytic polypeptide or enzyme. Any of thesemethods can be found in Sambrook, et al. or in CURRENT PROTOCOLS INMOLECULAR BIOLOGY, F. Ausubel, et al., ed. Greene Publishing andWiley-Interscience, New York (1987) (“Ausubel”).

A genomic library of the collected samples may be prepared usingstandard techniques known in the art. clones were screened forlipase/esterase activities by plating onto substrate-containing media,to give approximately 1000 colonies per 7 cm² diameter plate. Positiveclones were identified by the faint zone of clearing around them.

Positive clones may then have the plasmid DNA sequenced, including theinserted DNA, using primer sites within the plasmid using standardtechniques. Complete coverage of the sequence was obtained by ‘primerwalking’ from both the 5′ and 3′ ends of the insert. See Kieleczawa etal., Science (1992) 258:1787-91.

The process for isolating a gene according to the second aspect of thepresent invention makes use of its homology to a nucleotide sequencecomprising all or part of the nucleotide sequence of encoding a novelpolypeptide or lipolytic enzyme. Examples of such processes include:

-   -   a) screening a gene library which presumably contains a        lipolytic polypeptide or enzyme gene using the nucleotide        sequence as a probe.    -   b) preparing a primer based on the nucleotide sequence        information, then performing PCR using a sample which presumably        contains a lipolytic polypeptide or enzyme gene as a template.

More specifically, process a) above comprises:

-   -   a) preparing a gene library which presumably contains a        lipolytic polypeptide or enzyme gene, screening the gene library        using a nucleotide sequence comprising all or part of the        nucleotide sequence of SEQ ID NO:2 as shown in the sequence        listing to select sequences which hybridize with the nucleotide        sequence comprising all or part of the nucleotide sequence of        SEQ ID NO:2 as shown in the sequence listing from the gene        library, then isolating the selected sequences, and isolating a        LIP2 gene from the sequences which have been selected and        isolated from the gene library.

The gene library may be a genomic DNA library or a cDNA library, and maybe prepared according to a known procedure.

To obtain high level expression of a cloned gene, the heterologous geneis preferably positioned about the same distance from the promoter as isin the naturally occurring lipolytic polypeptide or enzyme gene. As isknown in the art, however, some variation in this distance can beaccommodated without loss of promoter function.

Those skilled in the art are aware that a natural promoter can bemodified by replacement, substitution, addition or elimination of one ormore nucleotides without changing its function. The practice of theinvention encompasses and is not constrained by such alterations to thepromoter.

B. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding a novel lipolyticpolypeptide or enzyme may be incorporated into heterologous nucleic acidconstructs or vectors, capable of introduction into, and replication in,a bacterial, a filamentous fungal or yeast cell. The vectors and methodsdisclosed herein are suitable for use in host cells for the expressionof a lipolytic polypeptide or enzyme. Any vector may be used as long asit is replicable and viable in the cells into which it is introduced.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. Cloning and expressionvectors are also described in Sambrook et al., 1989, Ausubel F M et al.,1989, and Strathern et al., 1981, each of which is expresslyincorporated by reference herein. Appropriate expression vectors forfungi are described in van den Hondel, C. A. M. J. J. et al. (1991) In:Bennett, J. W. and Lasure, L. L. (eds.) More Gene Manipulations inFungi. Academic Press, pp. 396-428. The appropriate DNA sequence may beinserted into a plasmid or vector (collectively referred to herein as“vectors”) by a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) bystandard procedures. Such procedures and related sub-cloning proceduresare deemed to be within the scope of knowledge of those skilled in theart.

The expression vector/construct typically contains a transcription unitor expression cassette that contains all the additional elementsrequired for the expression of the heterologous sequence. A typicalexpression cassette thus contains a promoter operably linked to theheterologous nucleic acid sequence and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

The practice of the invention is not constrained by the choice ofpromoter in the genetic construct. The only constraint on the choice ofpromoter is that it is functional in the host cell used. A preferredpromoter when the transformation host cell is Bacillus is the aprEpromoter.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includebacteriophages λ and M13, as well as plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST,and LacZ. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, e.g., c-myc.

The elements that are typically included in expression vectors alsoinclude a replicon, a gene encoding antibiotic resistance to permitselection of bacteria that harbor recombinant plasmids, and uniquerestriction sites in nonessential regions of the plasmid to allowinsertion of heterologous sequences. The particular antibioticresistance gene chosen is not critical, any of the many resistance genesknown in the art are suitable.

The methods of transformation of the present invention may result in thestable integration of all or part of the transformation vector into thegenome of the host cell. However, transformation resulting in themaintenance of a self-replicating extra-chromosomal transformationvector is also contemplated.

The gene encoding the lipolytic polypeptide or enzyme of the presentinvention can be cloned using λ-phage (expression) vectors and E. colihost cells. (Alternatively PCR cloning using consensus primers designedon conserved domains may be used.) Applicants have discovered thattransformation of the gene encoding the lipolytic polypeptide or enzymeof the present invention and expression in E. coli results in an activeprotein. After a first cloning step in E. coli, a lipolytic polypeptideor enzyme gene according to the present invention can be transferred toa more preferred industrial expression host such as Bacillus orStreptomyces species, a filamentous fungus such as Aspergillus orTrichoderma, or a yeast such as Saccharomyces. High level expression andsecretion obtainable in these host organisms allows accumulation of thelipolytic polypeptide or enzyme in the fermentation medium from which itcan subsequently be recovered.

III. Host Organisms

Suitable host organisms may be any microbe useful in industrialsettings, such as bacterial, fungi, yeast and the like.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota. The filamentous fungi are characterized byvegetative mycelium having a cell wall composed of chitin, glucan,chitosan, mannan, and other complex polysaccharides, with vegetativegrowth by hyphal elongation and carbon catabolism that is obligatelyaerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Trichoderma, e.g., Trichodermalongibrachiatum, Trichoderma viride, Trichoderma koningii, Trichodermaharzianum; Penicillium sp.; Humicola sp., including Humicola insolensand Humicola grisea; Chrysosporium sp., including C. lucknowense;Gliocladium sp.; Aspergillus sp.; Fusarium sp., Neurospora sp., Hypocreasp., and Emericella sp. As used herein, the term “Trichoderma” or“Trichoderma sp.” refers to any fungal strains which have previouslybeen classified as Trichoderma or are currently classified asTrichoderma.

Examples of parental cell lines which may be treated and/or modified forlipolytic polypeptide or enzyme expression include, but are not limitedto, filamentous fungal cells. Examples of appropriate primary cell typesfor use in practicing the invention include, but are not limited to,Aspergillus and Trichoderma.

In one embodiment, the filamentous fungal parent cell is an Aspergillusniger, Aspergillus awamori, Aspergillus aculeatus, or Aspergillusnidulans cell.

In another embodiment, the filamentous fungal parent cell is aTrichoderma reesei cell.

In a further embodiment, the filamentous fungal parent cell is aHypocrea jecorina cell. This cell was previously referred to as T.reesei.

In a further embodiment, the host cell is a gram negative Bacteria. Inone embodiment, the gram negative Bacteria is Escherichia coli (“E.coli”). Numerous E. coli strains are known, for example JM101 hsdS recA(CBS 155.87); and K12 DH1 (ATCC 33849). In another embodiment, the gramnegative Bacteria is Pseudomonas sp. In another embodiment, thePseudomonas sp. is Psuedomonas pseudoalcaligenes.

In a yet further embodiment, the host cell is a Bacillus sp. One type ofBacillus strain of interest is a cell of an alkalophilic Bacillus.Numerous alkalophilic Bacillus strains are known (U.S. Pat. No.5,217,878 and Aunstrup et al., Proc IV IFS: Ferment. Technol. Today,299-305 (1972)). Another type of Bacillus strain of interest is a cellof an industrial Bacillus strain. Examples of industrial Bacillusstrains are B. licheniformis, B. lentus, B. subtilis, and B.amyloliquefaciens. In another aspect, the Bacillus host strain may be B.licheniformis, B subtilis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.pumilus, B. thuringiensis, B. clausii, and B. megaterium. Particularlypreferred are B. subtilis cells. U.S. Pat. Nos. 5,264,366 and 4,760,025(RE 34,606) disclose various Bacillus host strains that may be used inaccordance with the invention.

An industrial strain may be a non-recombinant strain of a Bacillus sp.,a mutant of a naturally occurring strain or a recombinant strain.Preferably the host strain is a recombinant host strain wherein apolynucleotide encoding a lipolytic polypeptide or enzyme has beenintroduced into the host. A further preferred host strain is a Bacillussubtilis host strain and particularly a recombinant Bacillus subtilishost strain. Numerous B. subtilis strains are known, for example, 1A6(ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 through PB1758,PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100(ATCC 39,094), GX4931, PBT 110, and PEP 211strain. Hoch et al., (1973)Genetics, 73:215-228; U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544and EP-A-0134048).

The use of B. subtilis as an expression host is disclosed in Palva etal. Gene (1982) 19:81-87. Also see Fahnestock and Fischer. J. Bacteriol.(1986) 165:796-804; and Wang et al., Gene (1988) 69:39-47 fordescriptions of heterologous gene expression in B. subtilis.

IV. Molecular Biology II

A. Nucleic Acid Constructs/Expression Vectors.

A lipolytic polypeptide or enzyme coding sequence may be inserted into asuitable vector according to well-known recombinant techniques and usedto transform host cell capable of lipolytic polypeptide or enzymeexpression. Due to the inherent degeneracy of the genetic code, othernucleic acid sequences which encode substantially the same or afunctionally equivalent amino acid sequence may be used to clone andexpress a lipolytic polypeptide or enzyme. Therefore it is appreciatedthat such substitutions in the coding region fall within the sequencevariants covered by the present invention.

The present invention also includes recombinant nucleic acid constructscomprising one or more of the lipolytic polypeptide or enzyme-encodingnucleic acid sequences as described above. The constructs comprise avector, such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence fora lipolytic polypeptide or enzyme: (i) in isolation; (ii) in combinationwith additional coding sequences; such as fusion protein or signalpeptide coding sequences, where the lipolytic polypeptide or enzymecoding sequence is the dominant coding sequence; (iii) in combinationwith non-coding sequences, such as introns and control elements, such aspromoter and terminator elements or 5′ and/or 3′ untranslated regions,effective for expression of the coding sequence in a suitable host;and/or (iv) in a vector or host environment in which the lipolyticpolypeptide or enzyme coding sequence is a heterologous gene.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer a lipolytic polypeptide orenzyme-encoding nucleic acid sequence into a cell in vitro. Forlong-term, production of a lipolytic polypeptide or enzyme, stableexpression is preferred. It follows that any method effective togenerate stable transformants may be used in practicing the invention.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as promoter and termination sequences. The vectormay comprise regulatory sequences, including, for example, non-codingsequences, such as introns and control elements, i.e., promoter andterminator elements or 5′ and/or 3′ untranslated regions, effective forexpression of the coding sequence in host cells (and/or in a vector orhost cell environment in which a modified soluble protein antigen codingsequence is not normally expressed), operably linked to the codingsequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, many of which are commercially availableand/or are described in Sambrook, et al., (supra).

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook et al., 1989; Freshney, 1987; Ausubel, et al., 1993; andColigan et al., 1991.

B. Methods for Transforming a Host Cell

After DNA sequences that encode the lipolytic polypeptide or enzyme havebeen cloned into DNA constructs, the DNA is used to transformmicroorganisms.

Various methods may be employed for delivering an expression vector, DNAvector or construct described above into cells in vitro. Methods ofintroducing nucleic acids into cells for expression of heterologousnucleic acid sequences are also known to the ordinarily skilled artisan,including, but not limited to electroporation; nuclear microinjection ordirect microinjection into single cells; bacterial protoplast fusionwith intact cells; use of polycations, e.g., polybrene or polyornithine;membrane fusion with liposomes, lipofectamine or lipofection-mediatedtransfection; high velocity bombardment with DNA-coatedmicroprojectiles; incubation with calcium phosphate-DNA precipitate;DEAE-Dextran mediated transfection; infection with modified viralnucleic acids; Agrobacterium-mediated transfer of DNA; and the like. Inaddition, heterologous nucleic acid constructs comprising a lipolyticpolypeptide or enzyme-encoding nucleic acid sequence can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection.

The DNA construct will generally be functionally attached, i.e.,operably linked, to a promoter. The transformed host cell is then grownunder conditions so as to express the lipolytic polypeptide or enzyme.Subsequently, the lipolytic polypeptide or enzyme may be isolated. Itmay be desirable to have the lipolytic polypeptide or enzyme in asubstantially pure form. Similarly, it may be desirable to have thelipolytic polypeptide or enzyme in an essentially pure form.

It should be understood that the source of the lipolytic polypeptide orenzyme should be considered in determining the optimal expression host.Additionally, the skilled worker in the field will be capable ofselecting the best expression system for a particular gene throughroutine techniques utilizing the tools available in the art.

One skilled in the art is well aware of methods for introducingpolynucleotide sequences into E. coli cells, See for example, Andreoli,et al, U.S. Pat. No. 5,278,066. Some published methods for theintroduction of DNA constructs into lipase producing strains of E. coliinclude Sanchez, M, et al, Biotechnol. Bioeng. 78(3):339-45 (2002);Dartois, V, et al, Biochim. Biophys. Acta, 1131(3):253-260 (1992); andCho, A R, et al, EMS Microbiol Lett. 186(2):235-238 (2000), incorporatedby reference.

One skilled in the art is well aware of methods for introducingpolynucleotide sequences into Bacillus cells. See for example, Ferrariet al., Genetics pg 57-72 in Harwood et al. Ed. “Bacillus”, PlenumPublishing Corp. 1989 wherein methods of transformation, includingprotoplast transformation and congression; transduction; and protoplastfusion are disclosed. Methods of transformations are particularlypreferred to introduce a DNA construct according to the invention into ahost cell. In particular methods are also described for B. subtilis inChang et al., Mol. Gen. Genet. 168:11-115 (1979); for B. megaterium inVorobjeva et al., FEMS Microbiol. Letters 7:261-263 (1980); for Bamyloliquefaciens in Smith et al., Appl. and Env. Microbiol. 51:634(1986); for B. thuringiensis in Fisher et al., Arch. Microbiol.139:213-217 (1981); and for B. sphaericus in McDonald, J. Gen.Microbiol. 130:203 (1984). Reference is also made to Saunders et al., J.Bacteriol. 157:718-726 (1984); Hoch et al., J. Bacteriol. 93:1925-1937(1967); Mann et al., Current Microbiol. 13:131-135 (1986); and Holubova,Folia Microbiol. 30: 97 (1985).

A preferred general transformation and expression protocol for proteasedeleted Bacillus strains is provided in Ferrari et al., U.S. Pat. No.5,264,366, incorporated herein by reference. Transformation andexpression in Aspergillus is described in, for example, Berka et al.,U.S. Pat. No. 5,364,770, incorporated herein by reference.

Many standard transfection methods can be used to produce Trichodermareesei cell lines that express large quantities of the heterologusprotein. Some of the published methods for the introduction of DNAconstructs into cellulase-producing strains of Trichoderma includeLorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356;Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174;Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6:155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl.Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila andKolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, forStreptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich,UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi,1990, FEMS Microbiol. Lett. 55: 135-138), all incorporated herein byreference, and may be adapted for the introduction of a DNA constructencoding a lipolytic polypeptide or enzyme.

However, any of the well-known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, biolistics, liposomes, microinjection, plasma vectors,viral vectors and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). Also ofuse is the Agrobacterium-mediated transfection method described in U.S.Pat. No. 6,255,115. It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one gene into the host cell capable of expressing the heterologousgene.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofgenes under control of the promoter sequences. Large batches oftransformed cells can be cultured as described below. Finally, productis recovered from the culture using standard techniques.

Thus, the invention herein provides for the expression and enhancedsecretion of the inventive lipolytic polypeptides or enzyme whoseexpression is under control of promoter sequences, fusion DNA sequences,and various heterologous constructs. The invention also providesprocesses for expressing and secreting high levels of the inventivelipolytic polypeptides or enzyme(s).

C. Methods for Expressing a Lipolytic Enzyme

The methods of the invention rely on the use cells to express alipolytic polypeptide or enzyme, with no particular method of expressionrequired.

The invention provides host cells that have been transduced, transformedor transfected with an expression vector comprising a lipolyticpolypeptide or enzyme-encoding nucleic acid sequence. The cultureconditions, such as temperature, pH and the like, are those previouslyused for the parental host cell prior to transduction, transformation ortransfection and will be apparent to those skilled in the art.

In one approach, a bacterial, fungal or yeast cell is transfected withan expression vector having a promoter or biologically active promoterfragment or one or more (e.g., a series) of enhancers which functions inthe host cell line, operably linked to a DNA segment encoding alipolytic polypeptide or enzyme, such that the lipolytic polypeptide orenzyme is expressed in the cell line.

Thus, the present invention provides host cells comprising cells whichhave been modified, selected and cultured in a manner effective toresult in lipolytic polypeptide or enzyme production or expressionrelative to the corresponding non-transformed parental cell.

Host cells expressing a lipolytic polypeptide or enzyme are culturedunder conditions typically employed to culture the parental cell line.Generally, cells are cultured in a standard medium containingphysiological salts and nutrients, such as described in Pourquie, J. etal., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J.P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., Appl.Environ. Microbiol. 63:1298-1306, 1997. Culture conditions are alsostandard, e.g., cultures are incubated at 28° C. in shaker cultures orfermenters until desired levels of lipolytic polypeptide or enzymeexpression are achieved.

Preferred culture conditions for a given host cell may be found in thescientific literature and/or from the source of the fungi such as theAmerican Type Culture Collection (ATCC; <www.atcc.org>). After cellgrowth has been established, the cells are exposed to conditionseffective to cause or permit the expression of a lipolytic polypeptideor enzyme.

A selectable marker must be chosen so as to enable detection of thetransformed microorganism. Any selectable marker gene that is expressedin the selected microorganism will be suitable. For example, withAspergillus sp., the selectable marker is chosen so that the presence ofthe selectable marker in the transformants will not significantly affectthe properties thereof. Such a selectable marker may be a gene thatencodes an assayable product. For example, a functional copy of anAspergillus sp. gene may be used which if lacking in the host strainresults in the host strain displaying an auxotrophic phenotype.

Although the following discusses the Aspergillus system, similarprocedures for Trichoderma and other fungal systems, as well asbacterial systems, may be used as will be appreciated by one skilled inthe art.

DNA encoding the lipolytic polypeptide or enzyme is then prepared forinsertion into an appropriate microorganism. According to the presentinvention, DNA encoding a lipolytic polypeptide or enzyme comprises theDNA necessary to encode for a protein that has functional lipolyticactivity. The DNA fragment encoding the lipolytic polypeptide or enzymemay be functionally attached to a promoter sequence, for example, thefungal promoter of the glaA gene or the bacterial promoter of the aprEgene.

It is also contemplated that more than one copy of DNA encoding alipolytic polypeptide or enzyme may be recombined into the strain tofacilitate overexpression. The DNA encoding the lipolytic polypeptide orenzyme may be prepared by the construction of an expression vectorcarrying the DNA encoding the lipolytic polypeptide or enzyme. Theexpression vector carrying the inserted DNA fragment encoding thelipolytic polypeptide or enzyme may be any vector which is capable ofreplicating autonomously in a given host organism or of integrating intothe DNA of the host, typically a plasmid. In preferred embodiments twotypes of expression vectors for obtaining expression of genes arecontemplated. The first contains DNA sequences in which the promoter,gene-coding region, and terminator sequence all originate from the geneto be expressed. Gene truncation may be obtained where desired bydeleting undesired DNA sequences (e.g., coding for unwanted domains) toleave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences. A selectablemarker is also contained on the vector allowing the selection forintegration into the host of multiple copies of the novel genesequences.

The second type of expression vector is preassembled and containssequences required for high-level transcription and a selectable marker.It is contemplated that the coding region for a gene or part thereof canbe inserted into this general-purpose expression vector such that it isunder the transcriptional control of the expression cassettes promoterand terminator sequences. For example, pRAX is such a general-purposefungal expression vector. Genes or part thereof can be inserteddownstream of the strong glaA promoter. An example of an integrativeexpression vector is the pTrex vector. Genes or part thereof can beinserted downstream of the strong cbh1 promoter.

In the vector, the DNA sequence encoding the lipolytic polypeptide orenzyme of the present invention should be operably linked totranscriptional and translational sequences, i.e., a suitable promotersequence and signal sequence in reading frame to the structural gene.The promoter may be any DNA sequence that shows transcriptional activityin the host cell and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. An optional signal peptideprovides for extracellular production of the lipolytic polypeptide orenzyme. The DNA encoding the signal sequence is preferably that which isnaturally associated with the gene to be expressed, however the signalsequence from any suitable source is contemplated in the presentinvention.

The procedures used to fuse the DNA sequences coding for the lipolyticpolypeptide or enzyme of the present invention with the promoter intosuitable vectors are well known in the art.

D. Methods of Analysis For Lipolytic Enzyme Nucleic Acid CodingSequences and/or Protein Expression.

In order to evaluate the expression of a lipolytic polypeptide or enzymeby a cell line that has been transformed with a lipolytic polypeptide orenzyme-encoding nucleic acid construct, assays can be carried out at theprotein level, the RNA level or by use of functional bioassaysparticular to lipolytic activity and/or lipolytic polypeptide or enzymeproduction.

In general, assays employed to analyze the expression of a lipolyticpolypeptide or enzyme include, Northern blotting, dot blotting (DNA orRNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction),or in situ hybridization, using an appropriately labeled probe (based onthe nucleic acid coding sequence) and conventional Southern blotting andautoradiography.

In addition, the production and/or expression of a lipolytic polypeptideor enzyme may be measured in a sample directly, for example, by assaysfor lipolytic activity, expression and/or production. Such assays aredescribed, for example, See, for example, Gupta et al, Biotechnol. Appl.Biochem. (2003) 37:63-71, Andre, Christophe, et al, U.S. Pat. No.5,990,069 (International Publication WO 96/18729A1). which are expresslyincorporated by reference herein.

In addition, protein expression, may be evaluated by immunologicalmethods, such as immunohistochemical staining of cells, tissue sectionsor immunoassay of tissue culture medium, e.g., by Western blot or ELISA.Such immunoassays can be used to qualitatively and quantitativelyevaluate expression of a lipolytic polypeptide or enzyme. The details ofsuch methods are known to those of skill in the art and many reagentsfor practicing such methods are commercially available.

V. Protein Expression

Proteins of the present invention are produced by culturing cellstransformed with an expression vector containing the inventive lipolyticpolypeptide or enzyme gene whose expression is under control of promotersequences. The present invention is particularly useful for enhancingthe intracellular and/or extracellular production of polypeptides orenzymes. The polypeptides or enzymes may be homologous or heterologous.

Polypeptides or enzymes of the present invention may also be modified ina way to form chimeric molecules comprising a polypeptide or enzyme ofinterest fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof the polypeptide or enzyme of interest with a tag polypeptide whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino- or carboxyl-terminusof the polypeptide or enzyme of interest.

Various tag polypeptides/enzymes and their respective antibodies arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelationtags, the flu HA tag polypeptide and its antibody 12CA5 (Field et al.,Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular andCellular Biology 5:3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6):547-553 (1990)). Other tag polypeptides include theFLAG-peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3epitope peptide (Martin et al., Science 255:192-194 (1992)); tubulinepitope peptide (Skinner et al., J. Biol. Chem. 266:15163-15166 (1991));and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc.Natl. Acad. Sci. USA 87:6393-6397 (1990)).

Conditions appropriate for expression of said LIP2 gene comprisesproviding to the culture the components necessary for growth and/orexpression of the inventive lipolytic polypeptide or enzyme. Optimalconditions for the production of the polypeptides or enzymes will varywith the choice of the host cell, and with the choice of protein to beexpressed. Such conditions will be easily ascertained by one skilled inthe art through routine experimentation or optimization.

E. Methods for Purifying a Lipolytic Enzyme

In general, a lipolytic polypeptide or enzyme produced in cell cultureis secreted into the medium and may be purified or isolated, e.g., byremoving unwanted components from the cell culture medium. However, insome cases, a lipolytic polypeptide or enzyme may be produced in acellular form necessitating recovery from a cell lysate. In such casesthe lipolytic polypeptide or enzyme is purified from the cells in whichit was produced using techniques routinely employed by those of skill inthe art. Examples include, but are not limited to, affinitychromatography (Tilbeurgh et al., 1984), ion-exchange chromatographicmethods (Goyal et al., 1991; Fliess et al., 1983; Bhikhabhai et al.,1984; Ellouz et al., 1987), including ion-exchange using materials withhigh resolution power (Medve et al., 1998), hydrophobic interactionchromatography (Tomaz and Queiroz, 1999), and two-phase partitioning(Brumbauer, et al., 1999).

The polypeptide or enzyme of interest is typically purified or isolatedafter expression. The polypeptide or enzyme of interest may be isolatedor purified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample.

Typically, the lipolytic polypeptide or enzyme is fractionated tosegregate polypeptides or enzymes having selected properties, such asbinding affinity to particular binding agents, e.g., antibodies orreceptors; or which have a selected molecular weight range, or range ofisoelectric points.

Once expression of a given lipolytic polypeptide or enzyme is achieved,the lipolytic polypeptide or enzyme thereby produced is purified fromthe cells or cell culture. Exemplary procedures suitable for suchpurification include the following: antibody-affinity columnchromatography, ion exchange chromatography; ethanol precipitation;reverse phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; and gel filtration using, e.g., Sephadex G-75.

Standard purification methods include electrophoretic, molecular,immunological and chromatographic techniques, including ion exchange,hydrophobic, affinity, and reverse-phase HPLC chromatography, andchromatofocusing. For example, the polypeptide or enzyme of interest maybe purified using a standard anti-polypeptide or enzyme of interestantibody column. Ultrafiltration and diafiltration techniques, inconjunction with polypeptide or enzyme concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,Protein Purification (1982). The degree of purification necessary willvary depending on the use of the polypeptide or enzyme of interest. Insome instances no purification will be necessary.

VI. Utility of Lipolytic Enzymes

According to the invention, an inventive lipolytic polypeptide or enzymeof the invention may typically be a component of a detergentcomposition. As such, it may be included in the detergent composition inthe form of a non-dusting granulate, a stabilized liquid, or a protectedenzyme. Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) or4,689,297 (to Genencor Intl.) and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molecular weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. The inventive lipolytic polypeptides orenzymes may be encapsulated, for example, using the methods described inU.S. Pat. No. 6,420,333. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in patent GB1483591. Liquid enzyme preparations may, for example, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Other enzymestabilizers are well known in the art. Protected enzymes may be preparedaccording to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules, paste or liquid. The detergentcompositions according to the invention can be used depending on theirformulation as a washing powder, granule or liquid to wash fabrics; as aspot-removing product to remove spots or degrease objects or to removespots from fabric before cleaning; and as a powder or granular liquidfor dishwashing. A liquid detergent may be aqueous, typically containingup to 70% water and 0-30% organic solvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as an amylase, a pullulanase, a cutinase, a protease, acellulase, a peroxidase, an oxidase, (e.g. laccase) and/or anotherlipase.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzene-sulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative such as, e.g., an aromatic borateester, and the composition may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

A lipolytic polypeptide or enzyme of the invention may be incorporatedin concentrations conventionally employed in detergents. It is atpresent contemplated that, in a detergent composition of the invention,an inventive lipolytic polypeptide or enzyme may be added in an amountcorresponding to 0.00001-1 mg (calculated as pure enzyme protein) of thelipolytic polypeptide or enzyme per liter of wash liquor.

The detergent compositions of this invention may employ besides thelipolytic polypeptide or enzyme composition, a surfactant, includinganionic, non-ionic and ampholytic surfactants, a hydrolase, buildingagents, bleaching agents, bluing agents and fluorescent dyes, cakinginhibitors, solubilizers, cationic surfactants and the like. All ofthese components are known in the detergent art. The lipolyticpolypeptide or enzyme composition as described above can be added to thedetergent composition either in a liquid diluent, in granules, inemulsions, in gels, in pastes, and the like. Such forms are well knownto the skilled artisan. When a solid detergent composition is employed,the lipolytic polypeptide or enzyme composition is preferably formulatedas granules. Preferably, the granules can be formulated so as to containa lipolytic polypeptide or enzyme protecting agent.

Preferably the lipolytic polypeptide or enzyme compositions are employedfrom about 0.05 weight percent to about 5 weight percent relative to thetotal detergent composition. More preferably, the lipolytic polypeptideor enzyme compositions are employed from about 0.2 weight percent toabout 5 weight percent relative to the total detergent composition.

In addition the desired lipolytic polypeptide or enzyme nucleic acidsequence finds utility in the identification and characterization ofrelated nucleic acid sequences. A number of techniques useful fordetermining (predicting or confirming) the function of related genes orgene products include, but are not limited to, (A) DNA/RNA analysis,such as (1) overexpression, ectopic expression, and expression in otherspecies; (2) gene knock-out (reverse genetics, targeted knock-out, viralinduced gene silencing (VIGS, see Baulcombe, 1999); (3) analysis of themethylation status of the gene, especially flanking regulatory regions;and (4) in situ hybridization; (B) gene product analysis such as (1)recombinant protein expression; (2) antisera production, (3)immunolocalization; (4) biochemical assays for catalytic or otheractivity; (5) phosphorylation status; and (6) interaction with otherproteins via yeast two-hybrid analysis; (C) pathway analysis, such asplacing a gene or gene product within a particular biochemical orsignaling pathway based on its overexpression phenotype or by sequencehomology with related genes; and (D) other analyses which may also beperformed to determine or confirm the participation of the isolated geneand its product in a particular metabolic or signaling pathway, and helpdetermine gene function.

A purified form of a lipolytic polypeptide or enzyme may be used toproduce either monoclonal or polyclonal antibodies specific to theexpressed protein for use in various immunoassays. (See, e.g., Hu etal., 1991). Exemplary assays include ELISA, competitive immunoassays,radioimmunoassays, Western blot, indirect immunofluorescent assays andthe like. In general, commercially available antibodies and/or kits maybe used for the quantitative immunoassay of the expression level oflipolytic polypeptides or enzymes.

EXAMPLES

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein.

Example 1 Sample Collection and Processing

This example illustrates how to collect samples and process them toobtain sufficient DNA to create a cDNA library.

Samples of water and sediment (250 ml) were collected from the littoralzone of Lake Elmenteita, Kenya using a 250-ml stainless steel beakermounted on the end of a flexible extendible 1-m pole and placed insealable plastic containers (Whirlpak) for transport to the laboratoryat ambient temperature. The pH of the samples was 10 to 10.5 with atemperature 23° C. to 27° C., and a conductivity of 10.69-11.00 mS cm⁻¹.

To collect the microbial flora, water (350-750 ml) from the lakes wasfiltered on site (using a hand operated vacuum pump) through a sequenceof sterile membrane filters (47 mm diameter), composed of cellulosenitrate or cellulose acetate, of decreasing pore size, until all waterflow stopped. The sequence of filters was 8 μm, 3 μm and 0.22 μm. Theindividual membrane filters were placed immediately into 10 ml of cold,sterile cell stabilization buffer (TES) containing 10 mM Tris HCl,pH8.0; 1 mM EDTA and 5% w/v NaCl in 30 ml sterile plastic universaltubes and kept on ice in a refrigerated cool box until they could beprocessed further, usually within 4 hours of sampling. The microbialmaterial on the filters was dispersed by vigorous vortex mixing withsterile glass beads (5 ml) and the cells pelleted in microfuge tubes bycentrifugation at 13,000 g for 5 min. The microbial material wasaliquoted to the microfuge tubes in volumes estimated to contain theequivalent of 10⁸ to 10⁹ bacterial cells, giving a total of 12 tubes.The DNA was extracted using the GenomicPrep™ Cells and Tissue DNAisolation kit (Amersham Pharmacia biotech, Piscataway, N.J., USA)following the manufacturer's instructions. Cells in each tube wereresuspended in 600 μl of the Cell Lysis Solution provided, and incubatedat 80° C. for 5 min to lyse the cells. Samples prepared by this methodare stable at room temperature for at least 18 months, and weretransported back to the laboratory in this form. DNA extraction wascompleted by RNase A treatment, protein precipitation and isopropanolprecipitation of the DNA following the manufacturer's protocol. Each DNApellet was dissolved in 100 μl sterile Tris buffer 10 mM pH 8.5.

DNA yield was estimated by running 5 μl samples on a 0.5% w/v agarosegel and comparing with known amounts of bacterial genomic DNA. Thesamples were pooled, giving a total of about 20 μg DNA. When loweryields were encountered, the material was supplemented with about 30-50%extra DNA extracted from the water samples which were collected at thesame time as the on-site material and stored at 4° C. in the laboratoryuntil required. In this case the microbial mass was pelleted bycentrifugation. DNA isolation was carried out as described above, exceptthat incubation with 50 μl lysozyme solution (50 mg ml⁻¹ in 10 mMTris-HCl pH 8, 1 mM EDTA) for 30 minutes at 37° C. preceded the additionof the lysis solution, in order to degrade Gram positive cell walls.About 30 μg DNA was the amount of starting material that preliminaryexperiments had shown was needed to carry out the trial and bulkrestriction digestion and size fractionation to give sufficient materialfor library construction.

Selective Enrichment Culture

Selective enrichment culture from Lake Elmenteita was prepared by adding1 ml of pooled samples of water and sediment to 200 ml of a sterilealkaline minimal medium. The lipase selective medium contained, in g perliter, 1 g Yeast extract (Difco), 1 g K₂HPO₄, 0.2 g MgSO₄.7H₂O, 10 gNa₂CO₃, 40 g NaCl and 10% v/v olive oil. Cultures were grown at 37° C.for 3-5 days until bacterial growth was clearly visible. Cells wereobtained by centrifugation and DNA was extracted according to the methodof Pitcher et al., (Pitcher D G, Saunders N A, Owen R J (1989). Rapidextraction of bacterial genomic DNA with guanidium thiocyanate. Lettersin Applied Microbiology 8: 151-156).

Example 2 Library Construction

The following example details how to prepare a DNA library for use inscreening and detection of novel sequences in E. coli.

Preparation of DNA

The pooled DNA was used for construction of the genomic DNA library. Thepurified DNA was partially digested with Sau3A1 to give an averagefragment size in the range 2-10 kb. Restricted DNA was size fractionatedby electrophoresis on 0.5% agarose in TAE (0.04M Tris-acetate, 0.001MEDTA pH 8.0). Material in the 2 to 10 kb range was excised and replacedin a well of the same size cut in an unused part of the agarose gel andconcentrated to a narrow band by reversed electrical current. The DNAband was excised and DNA extracted using the QIAGEN (Crawley, UK)QIAEXII gel extraction kit, following the manufacturer's guidelines. Theeluted DNA was precipitated with ethanol and resuspended in 10 mM TrisHCl buffer, pH 8.5.

Preparation of Lambda Libraries

The restricted DNA was cloned into a Lambda vector using theZAP-Express™ vector kit (predigested with BamH1 and alkaline phosphatasetreated) and the Gigapak® III Gold packaging extract (Stratagene,Amsterdam, The Netherlands) following the manufacturer's protocol. Theprimary libraries were amplified as per protocol by plating aliquotscontaining ˜5×10⁴ pfu with host E. coli strain XL1-Blue MRF′ on 150 mmPetri dishes and eluting the phage in buffer. Amplified libraries werestored in 7% v/v dimethyl sulphoxide at −80° C. after freezing in liquidnitrogen. Primary titres were generally ≧10⁶ pfu, and afteramplification≧10⁹ pfu ml⁻¹.

Assessment of Library Quality

The phagemid vector pBK-CMV was excised from the Lambda ZAP libraryusing ExAssist helper phage (Stratagene) as described by themanufacturer, and used to infect E. coli strain XLOLR.Plasmid-containing clones were isolated by plating on Luria-Bertani (LB)agar containing 50 μg ml⁻¹ kanamycin. Blue:white screening in thepresence of Xgal [5-bromo-4-chloro-3-indoyl-β-D-galactoside] and IPTG[isopropylthio-β-D-galactoside] was used to determine cloningefficiency. If no DNA has been cloned into the Lambda vector, theβ-galactosidase gene is expressed in the presence of the inducer IPTG,resulting in cleavage of the substrate analogue Xgal to produce a bluepigment in the colony. If however a fragment of the genomic DNA has beensuccessfully cloned into the Lambda vector it disrupts the gene so thatno enzyme is produced and the colony remains white. The ratio of blue towhite colonies therefore can be used to calculate the percentage ofclones containing an insert. Twenty four colonies were selected atrandom and plasmid DNA prepared using the Wizard®Plus SV Miniprep DNApurification system (Promega UK, Southampton) Restriction analysis usingPst1 and HindIII which flank the BamH1 cloning site followed by agarosegel electrophoresis was used to determine insert sizes>90% of the clonescontained inserts in the 2-10 Kb size range.

Example 3 Library Screening for Esterase/Lipase

DNA libraries in the pBK-CMV phagemid were screened for esterase/lipaseactivity in a plate assay of the E. coli clones. To detectesterase/lipase activity the genomic libraries were plated on LB agarcontaining kanamycin (50 μml⁻¹), and after growth replica-plated ontotributyrin agar (Oxoid) containing IPTG (15 μl of a 0.5 M solutionspread on the surface of the agar in a 7 cm diameter Petri dish).Positive clones were identified by the faint zone of clearing aroundthem.

The sample from which the Lake Elmenteita environmental genomic librarywas made was enriched for microorganisms able to grow in the presenceolive oil as a sole carbon source. A library of genomic DNA was madefrom this minimal olive oil enrichment culture and yielded two positiveclones after screening 60,000 colonies for esterase activity ontributyrin agar, see Table 1. Agarose gel electrophoresis afterrestriction enzyme digestion showed that one, LIP2, was contained in acloned insert of about 2.5 kb in size and the other, LIP2, in a clonedinsert of about 3.5 kb.

Sequencing by primer walking indicated a LIP2 insert sequence of 3112 bpin length. Three main ORFs were identified, with the most probablelipase/esterase encoding region being 645 bp, encoding a predictedprotein 214amino acids long, see FIG. 4. It had highest homology of 43%over 197amino acids in a BLASTp search to an acyl-coA thioesterase Iprecursor of E. coli, U082664. A second putative ORF of 242amino acidshas 34% identity over 230amino acids to a probable short-chaindehydrogenase from Pseudomonas aeruginosa. (NP_(—)252127.1). The thirdORF of 187amino acids has 37% identity to a2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase fromXylella fastidiosa (NP_(—)298745.1, pir A82680) over 94amino acids.

Table 1 summarises the libraries from which enzyme activities describedhere have been discovered, including the names of the library, incidenceof positive clones, name of clone, size of cloned insert, size ofpredicted protein and identity to proteins present in the data bases.

TABLE 1 Details of the esterase/lipase clone libraries Incidence Name ofCloned Library of positive clones insert screened clones sequenced sizebp ORF Information Lake Elmenteita 1/100,000 ELIP 4313 1209 bp, 402aa,environmental 67% identity S. typhimurium NP_460582.1 Lake Elmenteita1/30,000 LIP1 2285 792 bp, 263aa, selective olive 42% identity oilenrichment V. cholerae NP_232345. LIP2 3112 645 bp, 214aa 43% identityE. coli U82664

The incidence of positive clones (Table 1) demonstrates that biasing anenvironmental sample to express a particular enzyme activity byenrichment culture does indeed result in an increased frequency forisolating that enzyme activity by more than 3-fold.

Example 4 Characterisation of the Esterase/Lipase-Positive Clones

Plasmid DNA was isolated from three esterase/lipase-positive clones, andthe size of the inserts determined by restriction digestion as describedabove. DNA sequencing of the plasmid DNA (using primer sites in thepBKCMV plasmid) was carried out by the Protein and Nucleic AcidChemistry Laboratory at Leicester University, using the Perkin Elmer‘BigDye’ terminator chemistry and the model 377 ABI automated DNAsequencer. Complete coverage of the sequence was obtained by ‘primerwalking’ from both the 5′ and 3′ ends of the insert. The sequence wasedited using Applied Biosystems multisequence editor Seqed™ version1.0.3. Sequence was assembled with programmes in the GCG WisconsinPackage, version 10.2-UNIX, available at the University of Leicester.Comparison of sequences to those in the databases was made using BLASTX2.1.3 and ORF finder was used to identify possible open reading frames.The nucleotide sequence of the inserted environmental DNA in aesterase/lipase-positive clone is shown in FIG. 1.

Comparison of the inserted environmental DNA sequence to proteinsequences in the databases was made using the BLASTx program.

Example 5 Identification of Esterase/Lipase Genes

Possible Open Reading Frames (ORF) in the nucleotide sequence of theinserted environmental DNA of 3 clones were identified using the ORFFind facility of the MapDraw program (DNASTAR, Brighton, Mass., USA) orORF Search from the Vector NTI Suite of programs (InforMax®, NorthBethesda, Md., USA). The results are recorded in FIG. 4.

The identified ORF's were examined by BLAST programs. The coding regionfor LIP2 is shown in FIG. 2.

An examination of the ORF nucleotide sequences using the BLASTx program,which compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase, revealed surprisingly, very low similarity (42-67%) to anumber of putative bacterial esterases. It is very probable that enzymeswith homology this low would not have been detected using conventionmethods using DNA probes based on known esterase/lipase gene sequences,especially given the very high diversity of esterases/lipases alreadycharacterised.

The translated protein sequence of the esterase/lipase coding region isshown in FIG. 3.

LIP2 is most related to an acyl-coA thioesterase I precursor of E. coliwhich belongs to the GSDL family of esterases (C. Upton and J. T.Buckley (1995). A new family of lipolytic enzymes? Trends in BiochemicalSciences, 20: 178-179). LIP2 contains the sequence LVVLGDSLSAG whichbelongs to the degenerate sequence motif characteristic of this family,[LIVMFYAG]-[LIVMFYAG]-[LIVMFYAG]-[LIVMFYAG]-G-D-S-[LIVM]-x(1,2)-[TAG]-G.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

The invention claimed is:
 1. A substantially pure polypeptide, whereinsaid polypeptide comprises an amino acid sequence having at least 85%amino acid sequence identity with the amino acid sequence set forth inSEQ ID NO:3.
 2. The polypeptide of claim 1, wherein said polypeptidecomprises an amino acid sequence having at least 90% sequence identitywith the amino acid sequence set forth in SEQ ID NO:3.
 3. Thepolypeptide of claim 1, wherein said polypeptide comprises an amino acidsequence having at least 95% sequence identity with the amino acidsequence set forth in SEQ ID NO:3.
 4. A detergent composition comprisinga recombinant polypeptide according to claim 1.